First published online March 31, 2006; 10.1104/pp.106.079533
Plant Physiology 141:196-207 (2006)
© 2006 American Society of Plant Biologists
SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION
Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach1
Anne Endler,
Stefan Meyer,
Silvia Schelbert,
Thomas Schneider,
Winfriede Weschke,
Shaun W. Peters,
Felix Keller,
Sacha Baginsky,
Enrico Martinoia and
Ulrike G. Schmidt*
University of Zurich, Institute of Plant Biology, CH–8008 Zurich, Switzerland (A.E., S.M., S.S., T.S., S.W.P., F.K., E.M., U.G.S.); Institute of Plant Genetics and Crop Plant Research, D–06466 Gatersleben, Germany (W.W.); and Swiss Federal Institute of Technology, Institute of Plant Science and Functional Genomics Center Zurich, CH–8092 Zurich, Switzerland (S.B.)
The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H+-ATPases and H+-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.
1 This work was supported by the Plant Science Center Zurich-Basel (graduate research fellowship), by the project Novel Ion Channels in Plants (grant no. EU HPRN–CT–00245), and by the Deutsche Forschungsgemeinschaft (project no. ME 1955/2).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ulrike G. Schmidt (ulrike.schmidt{at}botinst.unizh.ch).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079533.
* Corresponding author; e-mail ulrike.schmidt{at}botinst.unizh.ch; fax 41–44–6348204.
Received February 22, 2006;
returned for revision February 22, 2006;
accepted March 16, 2006.
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